Pr13.5 promoter for robust t-cell and antibody responses

ABSTRACT

The invention encompasses recombinant poxviruses, preferably modified Vaccinia Ankara (MVA) viruses, comprising a Pr13.5 promoter operably linked to a nucleotide sequence encoding an antigen and uses thereof. The invention is drawn to compositions and methods for the induction of strong CD8 T cell and antibody responses to a specific antigen(s) by administering one or more immunizations of the recombinant MVA to a mammal, preferably a human.

This application is a continuation application of U.S. application Ser.No. 14/437,939, now U.S. Pat. No. 9,828,414, which is a National Phaseapplication under 35 U.S.C. § 371 of International Application No.PCT/EP2013/003239, filed Oct. 28, 2013, and claims the benefit under 35U.S.C. § 119(e) of U.S. Provisional Patent Application 61/719,429 filedOct. 28, 2012, the disclosures of which are incorporated by referenceherein in their entirety.

BACKGROUND OF THE INVENTION

MVA originates from the dermal vaccinia strain Ankara (Chorioallantoisvaccinia Ankara (CVA) virus) that was maintained in the VaccinationInstitute, Ankara, Turkey for many years and used as the basis forvaccination of humans. However, due to the often severe post-vaccinalcomplications associated with vaccinia viruses (VACV), there wereseveral attempts to generate a more attenuated, safer smallpox vaccine.

During the period of 1960 to 1974, Prof. Anton Mayr succeeded inattenuating CVA by over 570 continuous passages in CEF cells (Mayr etal., 1975, Passage History: Abstammung, Eigenschaften and Verwendung desattenuierten Vaccinia-Stammes MVA. Infection 3: 6-14). As part of theearly development of MVA as a pre-smallpox vaccine, there were clinicaltrials using MVA-517 (corresponding to the 517th passage) in combinationwith Lister Elstree (Stickl, 1974, Smallpox vaccination and itsconsequences: first experiences with the highly attenuated smallpoxvaccine “MVA”. Prev. Med. 3(1): 97-101; Stickl and Hochstein-Mintzel,1971, Intracutaneous smallpox vaccination with a weak pathogenicvaccinia virus (“MVA virus”). Munch Med Wochenschr. 113: 1149-1153) insubjects at risk for adverse reactions from vaccinia. In 1976, MVAderived from MVA-571 seed stock (corresponding to the 571st passage) wasregistered in Germany as the primer vaccine in a two-stage parenteralsmallpox vaccination program. Subsequently, MVA-572 was used inapproximately 120,000 Caucasian individuals, the majority childrenbetween 1 and 3 years of age, with no reported severe side effects, eventhough many of the subjects were among the population with high risk ofcomplications associated with conventional vaccinia virus (Mayr et al.,1978, The smallpox vaccination strain MVA: marker, genetic structure,experience gained with the parenteral vaccination and behaviour inorganisms with a debilitated defence mechanism (author's transl).Zentralbl. Bacteriol. (B) 167: 375-390). MVA-572 was deposited at theEuropean Collection of Animal Cell Cultures, Vaccine Research andProduction Laboratory, Public Health Laboratory Service, Centre forApplied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP40JG, United Kingdom, as ECACC V9401 2707.

Being that many passages were used to attenuate MVA, there are a numberof different strains or isolates, depending on the passage number in CEFcells. All MVA strains originate from Dr. Mayr and most are derived fromMVA-572 that was used in Germany during the smallpox eradicationprogram, or MVA-575 that was extensively used as a veterinary vaccine.MVA-575 was deposited on Dec. 7, 2000, at the European Collection ofAnimal Cell Cultures (ECACC) with the deposition number V001 20707.

By serial propagation (more than 570 passages) of the CVA on primarychicken embryo fibroblasts, the attenuated CVA-virus MVA (modifiedvaccinia virus Ankara) was obtained. MVA was further passaged byBavarian Nordic and is designated MVA-BN. MVA, as well as MVA-BN, lacksapproximately 13% (26.5 kb from six major and multiple minor deletionsites) of the genome compared with ancestral CVA virus. The deletionsaffect a number of virulence and host range genes, as well as a largefragment of the gene coding for A-type inclusion protein (ATI) and agene coding for a structural protein directing mature virus particlesinto A-type inclusion bodies. A sample of MVA-BN was deposited on Aug.30, 2000, at the European Collection of Cell Cultures (ECACC) undernumber V00083008.

MVA-BN can attach to and enter human cells where virally-encoded genesare expressed very efficiently. However, assembly and release of progenyvirus does not occur. Preparations of MVA-BN and derivatives have beenadministered to many types of animals, and to more than 2000 humansubjects, including immunodeficient individuals. All vaccinations haveproven to be generally safe and well tolerated.

The perception from many different publications is that all MVA strainsare the same and represent a highly attenuated, safe, live viral vector.However, preclinical tests have revealed that MVA-BN demonstratessuperior attenuation and efficacy compared to other MVA strains (WO02/42480). The MVA variant strains MVA-BN as, e.g., deposited at ECACCunder number V00083008, have the capability of reproductive replicationin vitro in chicken embryo fibroblasts (CEF), but no capability ofreproductive replication in human cells in which MVA 575 or MVA 572 canreproductively replicate. For example, MVA-BN has no capability ofreproductive replication in the human keratinocyte cell line HaCaT, thehuman embryo kidney cell line 293, the human bone osteosarcoma cell line143B, and the human cervix adenocarcinoma cell line HeLa. Further,MVA-BN strains fail to replicate in a mouse model that is incapable ofproducing mature B and T cells, and as such is severely immunecompromised and highly susceptible to a replicating virus. An additionalor alternative property of MVA-BN strains is the ability to induce atleast substantially the same level of immunity in vaccinia virusprime/vaccinia virus boost regimes when compared to DNA-prime/vacciniavirus boost regimes.

The term “not capable of reproductive replication” is used in thepresent application as defined in WO 02/42480 and U.S. Pat. No.6,761,893, which are hereby incorporated by reference. Thus, the termapplies to a virus that has a virus amplification ratio at 4 days afterinfection of less than 1 using the assays described in U.S. Pat. No.6,761,893, which assays are hereby incorporated by reference. The“amplification ratio” of a virus is the ratio of virus produced from aninfected cell (Output) to the amount originally used to infect the cellsin the first place (Input). A ratio of “1” between Output and Inputdefines an amplification status wherein the amount of virus producedfrom the infected cells is the same as the amount initially used toinfect the cells.

MVA-BN or its derivatives are, according to one embodiment,characterized by inducing at least substantially the same level ofimmunity in vaccinia virus prime/vaccinia virus boost regimes whencompared to DNA-prime/vaccinia virus boost regimes. A vaccinia virus isregarded as inducing at least substantially the same level of immunityin vaccinia virus prime/vaccinia virus boost regimes when compared toDNA-prime/vaccinia virus boost regimes if the CTL response as measuredin one of the “assay 1” and “assay 2” as disclosed in WO 02/42480,preferably in both assays, is at least substantially the same invaccinia virus prime/vaccinia virus boost regimes when compared toDNA-prime/vaccinia virus boost regimes. More preferably, the CTLresponse after vaccinia virus prime/vaccinia virus boost administrationis higher in at least one of the assays, when compared toDNA-prime/vaccinia virus boost regimes. Most preferably, the CTLresponse is higher in both assays.

WO 02/42480 discloses how vaccinia viruses are obtained having theproperties of MVA-BN. The highly attenuated MVA-BN virus can be derived,e.g., by the further passage of a modified vaccinia virus Ankara (MVA),such as MVA-572 or MVA-575.

In summary, MVA-BN has been shown to have the highest attenuationprofile compared to other MVA strains and is safe even in severelyimmunocompromised animals.

Although MVA exhibits strongly attenuated replication in mammaliancells, its genes are efficiently transcribed, with the block in viralreplication being at the level of virus assembly and egress. (Sutter andMoss, 1992, Nonreplicating vaccinia vector efficiently expressesrecombinant genes. Proc. Natl. Acad. Sci. U.S.A 89: 10847-10851; Carrolland Moss, 1997, Host range and cytopathogenicity of the highlyattenuated MVA strain of vaccinia virus: propagation and generation ofrecombinant viruses in a nonhuman mammalian cell line. Virology 238:198-211.) Despite its high attenuation and reduced virulence, inpreclinical studies MVA-BN has been shown to elicit both humoral andcellular immune responses to VACV and to the products of heterologousgenes cloned into the MVA genome (Harrer et al., 2005, TherapeuticVaccination of HIV-1-infected patients on HAART with recombinant HIV-1nef-expressing MVA: safety, immunogenicity and influence on viral loadduring treatment interruption. Antiviral Therapy 10: 285-300; Cosma etal., 2003, Therapeutic vaccination with MVA-HIV-1 nef elicitsNefspecific T-helper cell responses in chronically HIV-1 infectedindividuals. Vaccine 22(1): 21-29; Di Nicola et al., 2003, Clinicalprotocol. Immunization of patients with malignant melanoma withautologous CD34(+) cell-derived dendritic cells transduced ex vivo witha recombinant replication-deficient vaccinia vector encoding the humantyrosinase gene: a phase I trial. Hum Gene Ther. 14(14): 1347-1 360; DiNicola et al., 2004, Boosting T cell-mediated immunity to tyrosinase byvaccinia virus-transduced, CD34(+)-derived dendritic cell vaccination: aphase I trial in metastatic melanoma. Clin Cancer Res. 10(16):5381-5390.)

MVA-BN and recombinant MVA-BN-based vaccines can be generated, passaged,produced and manufactured in CEF cells cultured in serum-free medium.Many recombinant MVA-BN variants have been characterized for preclinicaland clinical development. No differences in terms of the attenuation(lack of replication in human cell lines) or safety (preclinicaltoxicity or clinical studies) have been observed between MVA-BN, theviral vector backbone, and the various recombinant MVA-based vaccines.

Induction of a strong humoral and cellular immune response against aforeign gene product expressed by a VACV vector is hampered by the factthat the foreign gene product has to compete with all of the more than150 antigens of the VACV vector for recognition and induction ofspecific antibodies and T cells. The specific problem is theimmunodominance of vector CD8 T cell epitopes which prevents inductionof a strong CD8 T cell response against the foreign gene product. (Smithet al., Immunodominance of poxviral-specific CTL in a human trial ofrecombinant-modified vaccinia Ankara. J. Immunol. 175:8431-8437, 2005.)This applies to replicating VACV vectors such as Dryvax, as well as fornon-replicating vectors like NYVAC and MVA.

For expression of a recombinant antigen (“neoantigen”) by VACV, onlypoxvirus-specific promoters, but not common eukaryotic promoters, can beused. The reason for this is the specific biology of poxviruses whichreplicate in the cytoplasm and bring their own, cell-autonomoustranscriptional machinery with them that does not recognize typicaleukaryotic promoters.

The viral replication cycle is divided into two major phases, an earlyphase comprising the first two hours after infection before DNAreplication, and a late phase starting at the onset of viral DNAreplication at 2-4 hours after infection.

The late phase spans the rest of the viral replication cycle from ˜2-20h after infection until progeny virus is released from the infectedcell. There are a number of poxviral promoter types which aredistinguished and named by the time periods within the viral replicationcycle in which they are active, for example, early and late promoters.(See, e.g., Davison and Moss, J. Mol. Biol. 210:771-784, 1989; Davisonand Moss, J. Mol. Biol. 210:749-769, 1989; and Hirschmann et al.,Journal of Virology 64:6063-6069, 1990, all of which are herebyincorporated by reference.)

Whereas early promoters can also be active late in infection, activityof late promoters is confined to the late phase. A third class ofpromoters, named intermediate promoters, is active at the transition ofearly to late phase and is dependent on viral DNA replication. Thelatter also applies to late promoters, however, transcription fromintermediate promoters starts earlier than from typical late promotersand requires a different set of transcription factors.

It became increasingly clear over recent years that the choice of thetemporal class of poxviral promoter for neoantigen expression hasprofound effects on the strength and quality of the neoantigen-specificimmune response. It was shown that T cell responses against neoantigensexpressed under the control of a late promoter are weaker than thoseobtained with the same antigen under an early promoter. (Bronte et al.,Antigen expression by dendritic cells correlates with the therapeuticeffectiveness of a model recombinant poxvirus tumor vaccine. Proc. Natl.Acad. Sci. U.S. A 94:3183-3188, 1997. Coupar et al., Temporal regulationof influenza hemagglutinin expression in vaccinia virus recombinants andeffects on the immune response. Eur. J. Immunol. 16:1479-1487, 1986.)

Even more strikingly, it was recently shown that in repeated autologousimmunizations with VACV as well as with the replication-defective VACVvector MVA, CD8 T cell responses against antigens under an exclusivelylate promoter can fail completely. This failure resulted in an almostundetectable antigen-specific CD8 T cell response after the secondimmunization. (Kastenmuller et al., Cross-competition of CD8+ T cellsshapes the immunodominance hierarchy during boost vaccination. J. Exp.Med. 204:2187-2198, 2007.)

Thus, early expression of neoantigens by VACV vectors appears to becrucial for efficient neoantigen-specific CD8 T cell responses. It hasalso been shown that an early-expressed VACV vector antigen not onlycompetes with late expressed antigens but also with other early antigensfor immunodominance in the CD8 T cell response. (Kastenmuller et al.,2007.) The specific properties of the early portion of the poxviralpromoter might thus be very important for induction of aneoantigen-specific T cell response. Moreover, it is a commonly heldview and a general rule that higher amounts of antigen are beneficialfor induction of stronger antigen-specific immune responses (for thepoxvirus field, see for example Wyatt et al., Correlation ofimmunogenicities and in vitro expression levels of recombinant modifiedvaccinia virus Ankara HIV vaccines. Vaccine 26:486-493, 2008).

A promoter combining 4 early promoter elements and a late promoterelement from the ATI gene has been described previously (Funahashi etal., Increased expression in vivo and in vitro of foreign genes directedby A-type inclusion body hybrid promoters in recombinant vacciniaviruses. J. Virol. 65:5584-5588, 1991; Wyatt et al., Correlation ofimmunogenicities and in vitro expression levels of recombinant modifiedvaccinia virus Ankara HIV vaccines. Vaccine 26:486-493, 2008), and hasbeen shown to direct increased early expression of antigen. However, Tcell responses induced by an antigen driven by such a promoter have onlybeen analyzed after a single immunization and were not apparentlydifferent from those obtained with the classical Pr7.5K promoter in thissetting. (Funahashi et al., Increased expression in vivo and in vitro offoreign genes directed by A-type inclusion body hybrid promoters inrecombinant vaccinia viruses. J. Virol. 65:5584-5588, 1991.)

Jin et al. Arch. Virol. 138:315-330, 1994, reported the construction ofrecombinant VACV promoters consisting of a VACV ATI promoter combinedwith tandem repeats (2 to 38 copies) of a mutated Pr7.5 promoteroperably linked to the CAT gene. Up to 10 repetitions of the mutatedPr7.5 promoter were effective in increasing early gene expression.Further repetition appeared to be inhibitory. With all constructs, theamount of CAT protein produced in the presence of cytosine arabinoside(AraC) (i.e. when the viral replication cycle was arrested in the earlyphase) was less than one-tenth of the amount produced in the absence ofAraC (Jin et al. Arch. Virol. 138:315-330, 1994).

Recently, it was shown that repeated immunizations of mice withrecombinant MVA expressing OVA under the control of a hybrid early-latepromoter (pHyb) containing five copies of a strong early element led tosuperior acute and memory CD8 T-cell responses compared to those toPr7.5- and PrS-driven OVA. Baur et al., Journal of Virology, Vol. 84(17): 8743-8752 (2010). Moreover, OVA expressed under the control ofpHyb replaced the MVA-derived B8R protein as the immunodominant CD8T-cell antigen after three or more immunizations. Id.

Assarsson et al., P.N.A.S. 105: 2140-45, 2008, simultaneously measuredthe expression levels of 223 annotated vaccinia virus genes duringinfection and determined their kinetics using a genome tiling arrayapproach. They found that many genes in the WR strain of Vaccinia virushad high transcription rates. Assarsson et al. provided some examples ofhighly expressed genes: immediate-early, VACWR-059 (double-strandedRNA-binding protein) and VACWR-184 (unknown); early, VACWR-018(unknown); early/late, VACWR-131 (core protein); and late, VACWR-169(unknown). Assarsson et al. indicated that, because of theirexceptionally high expression levels, these genes might be of specialinterest for future investigations, but did not identify the promotersinitiating transcription of these genes.

Yang et al., P.N.A.S. 107:11513-11518, 2010, used deep RNA sequencing toanalyze vaccinia virus (VACV) transcriptomes at progressive timesfollowing infection. Before viral DNA replication, transcripts from 118VACV ORFs were detected; after replication, transcripts from 93additional ORFs were characterized. The high resolution permitteddetermination of the precise boundaries of many mRNAs includingread-through transcripts and location of mRNA start sites and adjacentpromoters.

Orubu et al, PLoS ONE 7(6):e40167, 2012, showed that potent earlypromoters that drive expression of non-functional or non-essential MVAopen reading frames (ORFs) can be harnessed for immunogenic expressionof recombinant antigen. Precise replacement of the MVA orthologs ofC11R, F11L, A44L and B8R with a model antigen positioned to use the sametranslation initiation codon allowed early transgene expression similarto or slightly greater than that achieved by the commonly-used p7.5 orshort synthetic promoters. The frequency of antigen-specific CD8+ Tcells induced in mice by single shot or adenovirus-prime, rMVA-boostvaccination were similarly equal or marginally enhanced using endogenouspromoters at their authentic genomic loci compared to the traditionalconstructs. The enhancement in immunogenicity observed using the C11R orF11L promoters compared with p7.5 was similar to that obtained with themH5 promoter compared with p7.5.

Strong T cell and antibody responses against antigens encoded byrecombinant poxviruses can improve vaccine efficacy. Consequently, aneed in the art exists for compositions and methods capable of achievingstrong T cell and antibody responses against antigens encoded byrecombinant poxviruses, such as MVA. The invention fulfills this need.

BRIEF SUMMARY OF THE INVENTION

The invention encompasses a recombinant modified Vaccinia Ankara (MVA)virus comprising a Pr13.5 promoter linked to a nucleotide sequenceencoding a neoantigen and uses thereof. In one embodiment, the inventionencompasses a method of inducing a robust CD8 T cell response against aneoantigen in mammal, preferably a human, comprising administering oneor more immunizations of the MVA virus to the mammal, including a human.

In various embodiments, the Pr13.5 promoter comprises at least 1 copy ofa nucleic acid sequence of at least 40 bases having at least 95%, 98%,or 100% identity with SEQ ID NO:1.

In various embodiments, the Pr13.5 promoter comprises at least 1 copy ofa second nucleotide sequence of at least 31 nucleotides that has atleast 95%, 98% or 100% identity with SEQ ID NO:1.

In various embodiments, the Pr13.5 promoter comprises 2 copies of anucleotide sequence of at least 40 nucleotides that has 100% identitywith SEQ ID NO:1.

In various embodiments, the Pr13.5 promoter comprises SEQ ID NO:2.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the upstream sequence of the MVA013.5L gene (SEQ IDNO:3). Sequences of the Pr13.5-short and Pr13.5-long promoters aregiven. Dashed line: Pr13.5-long (Pos. 15878-15755). Solid line:Pr13.5-short (Pos. 15808-15755). Underlined: ATG start codon of MVA013.5(Pos. 15703-15701). TAA stop codon of MVA014L (Pos. 15878-15856). Blackarrows from below: transcription start sites as defined by RACE PCR(Pos. 15767 and 15747). Grey arrows from top: transcription start sitesas defined by Yang et al., 2010, suppl. data. Boxed: core promoter asdefined by Yang et al., 2010, suppl. data (Pos. 15913-15899). Positionsaccording to GenBank DQ983238.1

FIG. 2 depicts the sequence and position of the Pr13.5-long andPr13.5-short promoters in the MVA genome (SEQ ID NO:3). There is a 44 bpsequence repeat (direct repeat) in the upstream sequence of the MVA013.5gene. Boxed: boxed is the 44 bp repeated sequence in the upstreamsequence of 13.5, which is separated by a 36 bp spacer. Dashed line:Pr13.5-long (Pos. 15878-15755). Solid line: Pr13.5-short (Pos.15808-15755). Underlined: ATG start codon of MVA013.5 (Pos.15703-15701). Positions according to GenBank DQ983238.1.

FIG. 3 depicts RT-qPCR measuring ovalbumin-mRNA from HeLa cells infectedwith the indicated constructs at the post infection time pointsindicated.

FIG. 4 depicts Ova protein expression measured by FACS as meanfluorescence intensity (MFI) from HeLa cells infected with the indicatedconstructs at the post infection time points indicated. The mean of thewt (no Ova gene included) at 399 MFI reflects the background of theassay.

FIG. 5 depicts the average ratio of Ova+/B8R+ cells from mice vaccinatedwith the indicated constructs after the first, second and thirdimmunizations.

FIG. 6 depicts the average ratio of Ova+/B8R+ T cell response of mice at10 weeks after the third immunization with the indicated constructs.

FIGS. 7A and 7B depict antibody production from the indicated constructsafter the first, second and third immunizations. A. Geometric mean titer(GMT) of antibodies. B. Ratio of GMT compared to PrS promoter. Thepromoters MVA50L+PrSSL and MVA170R+PrSSL are the MVA promoters of therespective genes fused at the 5′ side of the synthetic Short Strong Latepromoter PrSSL promoter directly upstream of the ATG of the ovalbumingene. (AATTTTTAATATATAA; SEQ ID NO:7; PCT WO 2010/060632 A1.)

FIGS. 8A-8F depict a BLAST alignment of the nucleotide sequences ofvarious poxvirus Pr13.5 promoters with SEQ ID NO:1. Identicalnucleotides are depicted by dots, missing nucleotides are depicted bydashes, and changes are indicated by letters.

FIGS. 9A-9D depict accession numbers and names for the sequences in thealignments in FIGS. 8A-8F.

DETAILED DESCRIPTION OF THE INVENTION

HeLa cells were infected with MVA-BN and RNA was prepared. Primersspecific for various MVA ORFs were generated and RACE-PCR (FirstChoice®RLM-RACE Kit, Life Technologies, Darmstadt, Germany) was used togenerate PCR products representative of the MVA RNAs encoding theseORFs. The PCR products were sequenced to identify the transcriptionstart sites. Based on this information, promoters were identified forthe transcription of mRNAs encoding these ORFs. The MVA promoters forthe following ORFs were inserted into MVA constructs to drive expressionof the ovalbumin (OVA) gene: MVA13.5 (CVA022; WR 018), MVA050L (E3L; WR059), MVA022L (K1L; WR 032), and MVA170R (B3R; WR 185).

HeLa cells were infected in vitro with the recombinant MVA viruses andovalbumin protein expression was examined by FACS analysis. No ovalbuminprotein expression was detected by FACS analysis for constructscontaining the MVA050L (E3L; WR 059), MVA022L (K1L; WR 032), and MVA170R(B3R; WR 185) promoters at 2, or even 4, hours after infection. Incontrast, high level ovalbumin expression was detected with the MVA13.5(CVA022; WR 018) promoter already after 2 hours.

A putative promoter core element for the MVA13.5L ORF was previouslyidentified in Yang et al., 2010, as containing a 15 nt core sequence,and an untranslated leader of 177 nt. However, the current studyindicated that the transcriptional start sites used by MVA13.5L ORF weredownstream of the start site identified by Yang et al. by more than 100nucleotides. Consequently, the MVA13.5 promoter identified by theinventors differs from the promoter core element identified by Yang etal.

The MVA13.5 promoter identified by the inventors contains a repeat ofover 40 nucleotides: TAAAAATAGAAACTATAATCATATAATAGTGTAGGTTGGTAGTA (SEQID NO:1). The repeated sequence can also be found in many otherpoxviruses, for example, horsepox virus, monkeypox virus, cowpox virus,variola virus, vaccinia virus, camelpox virus, rabbitpox virus,Ectromelia virus, and taterapox virus (FIGS. 8 and 9).

Two MVA constructs were generated with promoters containing one copy(MVA13.5 short; SEQ ID NO:1) or two copies (MVA13.5 long; SEQ ID NO:2)of the repeat driving expression of the ovalbumin (OVA) gene. High levelovalbumin expression was detected after infection of HeLa cells in vitrowith both of the constructs. (FIG. 4.)

Ovalbumin RNA expression directed by various promoters in infected HeLacells in vitro was measured at various time points by RT-qPCR. BothMVA13.5 short and MVA13.5 long showed high levels of early RNAexpression. (FIG. 3.) MVA13.5 long showed the highest levels of earlyprotein expression.

CD8 T cell responses against recombinantly expressed OVA under controlof the promoters PrS, Pr7.5 opt+spacer, Pr13.5 short and Pr13.5 longwere determined in mice after one, two, and three immunizations ofrecombinant MVA per mouse (FIG. 5-6.). The OVA-specific andB8R(viral)-specific CD8 T cell response was determined by assessing thenumber of CD8 T cells specifically binding to MHC class I hexamers. TheMHC class I dextramers were complexed with their respective H-2Kbbinding peptides, SIINFEKL (SEQ ID NO:4) for OVA or TSYKFESV (SEQ IDNO:5) for the viral B8R peptide.

The average ratio of OVA-specific to B8R-specific CD8 T cells wasapproximately 2.5 for MVA13.5-long after 3 immunizations. The other 3constructs showed an average ratio of less than 1. Thus, a reversal ofthe immunodominance hierarchy could be achieved by using the Pr13.5 longpromoter for expression of the neoantigen, but not by using the otherpromoters.

Antibody responses against recombinantly expressed OVA under control ofvarious promoters were determined in mice after one, two, and threeimmunizations of recombinant MVA per mouse. (FIG. 7A-B.) The antibodyresponse with MVA13.5 long was substantially higher than the responseusing a recombinant MVA with the PrS promoter. Thus, the use of thePr13.5 long promoter to drive neoantigen expression from MVA providesunexpectedly superior results.

Pr13.5 Promoters

The invention encompasses isolated nucleic acids comprising orconsisting of a Pr13.5 promoter. Within the context of this invention, a“Pr13.5 promoter” comprises at least 1 copy of a nucleic acid sequenceof at least 40 bases having at least 95% identity with SEQ ID NO:1.Thus, a “Pr13.5 promoter” can, in various embodiments, refer to an MVAnucleotide sequence, a synthetic sequence, or an analogous poxviralsequence from a poxvirus other than MVA. Preferably, the Pr13.5 promotercomprises at least 1 copy of a nucleic acid sequence of at least 40bases having at least 96%, 97%, 98%, 99%, or 100% identity with SEQ IDNO:1. The nucleic acid sequence is preferably 40, 41, 42, 43, 44, or 45bases in length.

The percent identity can be determined by visual inspection andmathematical calculation. Alternatively, the percent identity of twonucleic acid sequences can be determined by comparing sequenceinformation using the GAP computer program, version 6.0 described byDevereux et al. (Nucl. Acids Res. 12:387, 1984) and available from theUniversity of Wisconsin Genetics Computer Group (UWGCG). The preferreddefault parameters for the GAP program include: (1) a unary comparisonmatrix (containing a value of 1 for identities and 0 for non-identities)for nucleotides, and the weighted comparison matrix of Gribskov andBurgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz andDayhoff, eds., Atlas of Protein Sequence and Structure, NationalBiomedical Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0for each gap and an additional 0.10 penalty for each symbol in each gap;and (3) no penalty for end gaps. Other programs used by one skilled inthe art of sequence comparison may also be used.

Preferably, the Pr13.5 promoter is operably linked to a heterologousnucleic acid sequence. Within the context of this invention,“heterologous nucleic acid sequence” means a nucleic acid sequence towhich the promoter is not linked in nature. Within the context of thisinvention, “operably linked” means that the promoter can driveexpression of the heterologous nucleic acid sequence in a poxvirusinfected cell. The heterologous nucleic acid sequence preferably encodesa neoantigen. Within the context of this invention, a neoantigen refersto an antigen not naturally expressed by the poxviral vector.

The Pr13.5 promoter can be operably linked to a heterologous nucleicacid sequence by recombinant DNA technology. In various embodiments, theheterologous nucleic acid sequence is introduced into the 13.5 ORF ofthe poxvirus.

Preferably, the Pr13.5 promoter is a naturally occurring poxviruspromoter. For example, the Pr13.5 promoter can be from modified vacciniaAnkara (MVA) virus, monkeypox virus, cowpox virus, variola virus,vaccinia virus, camelpox virus, rabbitpox virus, Ectromelia virus, ortaterapox virus Pr13.5 promoter. Preferred Pr13.5 promoters can beselected from the viruses shown in FIG. 9 and the sequences shown inFIG. 8.

In various embodiments, the Pr13.5 promoter is a synthetic Pr13.5promoter.

The Pr13.5 promoter can contain 1, 2, 3, 4, 5, 6, or more copies of asequence of at least 40, 41, 42, 43, 44, or 45 nucleotides that has atleast 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:1.

Preferably, the Pr13.5 promoter contains 1 copy of the nucleotidesequence of SEQ ID NO:1.

In some embodiments, the Pr13.5 promoter contains 1 copy of thenucleotide sequence of SEQ ID NO:1 and 1, 2, 3, 4, 5, 6, or more copiesof a sequence of at least 40, 41, 42, 43, or 44 nucleotides that has atleast 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:1.

Preferably, the Pr13.5 promoter contains at least 1 copy of a nucleotidesequence of at least 40 bases that has at least 98% identity with SEQ IDNO:1.

In some embodiments, the Pr13.5 promoter contains 1 copy of a nucleotidesequence of at least 40 bases that has at least 95%, 96%, 97%, 98%, 99%,or 100%% identity with SEQ ID NO:1 and 1, 2, 3, 4, 5, 6, or more copiesof a second nucleotide sequence of at least 31 nucleotides that has atleast 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:1.Preferably the second nucleotide sequence is at least 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 bases.

Preferably, the repeated sequences are separated by 20-80 nucleotides,more preferably 30-40 nucleotides, and most preferably of 33, 35, 35,36, 37, 38, 39, or 40 nucleotides.

Preferably, the Pr13.5 promoter comprises at least one copy of thesequence:

(SEQ ID NO: 2) TAAAAATAGAAACTATAATCATATAATAGTGTAGGTTGGTAGTATTGCTCTTGTGACTAGAGACTTTAGTTAGGTACTGTAAAAATAGAAACTATAATCATATAATAGTGTAGGTTGGTAGTA.

In some embodiments, the Pr13.5 promoter comprises one or more of thenucleotide changes shown in FIG. 8.

The invention encompasses methods of expressing a neoantigen comprisingoperably linking a Pr13.5 promoter to a heterologous nucleic acidsequence.

Recombinant Poxviruses Comprising Pr13.5 Promoters

The invention encompasses a recombinant poxviral vector comprising aPr13.5 promoter operably linked to a heterologous nucleic acid sequence.In one embodiment, the heterologous nucleic acid sequence is insertedinto the 13.5 ORF of a poxvirus so as to operably link the heterologousnucleic acid sequence to the endogenous viral Pr13.5 promoter. Inanother embodiment, the heterologous nucleic acid sequence is linked toa Pr13.5 promoter and inserted into a site in the genome other than the13.5 ORF.

Preferably, the poxvirus vector is derived from poxviruses belonging tothe Chordopoxvirinae subfamily. Poxviruses include those belonging tothe genera Orthopoxvirus, Parapoxvirus, Avipoxvirus, Capripoxvirus,Lepripoxvirus, Suipoxvirus, Molluscipoxvirus and Yatapoxvirus. Mostpreferred are poxviruses belonging to the genera Orthopoxvirus andAvipoxvirus.

Other poxviruses such as racoonpox and mousepox may be employed in thepresent invention, for example, for the manufacture of wild-lifevaccine. Members of the capripoxvirus and leporipox are also includedherein as they may be useful as vectors for cattle and rabbits,respectively.

In other embodiments, the poxvirus is derived from avipoxviruses.Examples of avipoxviruses suitable for use in the present inventioninclude any avipoxvirus such as fowlpoxvirus, canarypoxvirus,uncopoxvirus, mynahpoxvirus, pigeonpoxvirus, psittacinepoxvirus,quailpoxvirus, peacockpoxvirus, penguinpoxvirus, sparrowpoxvirus,starlingpoxvirus and turkeypoxvirus. Preferred avipoxviruses arecanarypoxvirus and fowlpoxvirus.

Preferably, the poxvirus is a vaccinia virus, most preferably MVA. Theinvention encompasses recombinant MVA viruses generated with any and allMVA viruses. Preferred MVA viruses are MVA variant strains MVA-BN as,e.g., deposited at ECACC under number V00083008; MVA-575, deposited onDec. 7, 2000, at the European Collection of Animal Cell Cultures (ECACC)with the deposition number V001 20707; and MVA-572, deposited at theEuropean Collection of Animal Cell Cultures as ECACC V9401 2707.Derivatives of the deposited strain are also preferred.

Preferably, the MVA has the capability of reproductive replication invitro in chicken embryo fibroblasts (CEF) or other avian cell lines orin vivo in embryonated eggs, but no capability of reproductivereplication in human cells in which MVA 575 or MVA 572 canreproductively replicate. Most preferably, the MVA has no capability ofreproductive replication in the human keratinocyte cell line HaCaT, thehuman embryo kidney cell line 293, the human bone osteosarcoma cell line143B, and the human cervix adenocarcinoma cell line HeLa.

In preferred embodiments, the Modified vaccinia virus Ankara (MVA) virusis characterized by having the capability of reproductive replication invitro in chicken embryo fibroblasts (CEF) and by being more attenuatedthan MVA-575 in the human keratinocyte cell line HaCaT, in the humanbone osteosarcoma cell line 143B, and in the human cervix adenocarcinomacell line HeLa. Preferably, the MVA virus is capable of a replicationamplification ratio of greater than 500 in CEF cells.

Any antigen, including those that induce a T-cell response, can beexpressed by the recombinant MVA of the invention. Viral, bacterial,fungal, and cancer antigens are preferred. HIV-1 antigens, Dengue virusantigens, prostate-specific antigen (PSA) and prostatic acid phosphatase(PAP) antigen, HER-2/Neu antigens, anthrax antigens, measles virusantigens, influenza virus, picornavirus, coronavirus and respiratorysyncytial virus antigens are particularly preferred antigens.Preferably, the antigen is a foreign antigen or neoantigen.

The invention encompasses methods of making recombinant poxviruses,preferably MVA, comprising inserting a heterologous nucleic acidsequence into a poxvirus such that the heterologous nucleic acidsequence is operably linked to a Pr13.5 promoter.

The invention encompasses use of the recombinant poxviruses of theinvention in the manufacture of a medicament or vaccine for thetreatment or prevention of infections and diseases of a mammal,including a human.

The invention encompasses use of the recombinant poxviruses of theinvention for the treatment or prevention of infections and diseases ofa mammal, including a human.

The invention encompasses use of the recombinant poxviruses of theinvention as vaccines, particularly for the treatment or prevention ofinfections and diseases of a mammal, including a human.

Kits Comprising Recombinant MVA

The invention provides kits comprising the recombinant poxviral vector,preferably MVA virus, according to the present invention. The kit cancomprise at least one, two, three, four, or more containers or vials ofthe recombinant poxviral vector, preferably MVA virus, together withinstructions for the administration of the virus to a mammal, includinga human. The instructions can indicate that the recombinant virus isadministered to the mammal, preferably a human, in one or multiple(i.e., 2, 3, 4, 5, 6, etc.) dosages at specific timepoints (e.g., atleast 4 weeks, at least 6 weeks, at least 8 weeks after the previousadministration). Preferably, the instructions indicate that therecombinant virus is to be administered to a mammal, preferably a human,in at least 1, at least 2, at least 3, or at least 4 dosages.

Methods of Inducing a CD8 T Cell and/or Antibody Response

The invention encompasses methods of inducing a CD8 T cell and/orantibody response in a host. In preferred embodiments, the methodcomprises administering at least one, two, three, four, or fiveimmunizations of a recombinant poxvirus, preferably MVA, comprising aPr13.5 promoter to the mammal, including a human.

Administration to a Host

The recombinant poxvirus, preferably MVA, according to the invention canbe used for the treatment of a wide range of mammals including humansand even immune-compromised humans. Hence, the present invention alsoprovides a pharmaceutical composition and also a vaccine for inducing animmune response in a mammal, including a human.

The vaccine preferably comprises the recombinant poxvirus, preferablyMVA, in a concentration range of 10⁴ to 10⁹ TCID (tissue cultureinfectious dose) 50/ml, preferably in a concentration range of 10⁵ to5×10⁸ TCID₅₀/ml, more preferably in a concentration range of 10⁶ to 10⁸TCID₅₀/ml, and most preferably in a concentration range of 10⁷ to 10⁸TCID₅₀/ml, especially 10⁸ TCID₅₀/ml.

A preferred vaccination dose for mammal, preferably a human, comprises10⁶ to 10⁹ TCID₅₀, most preferably a dose of 10⁷ TCID₅₀ or 10⁸ TCID₅₀,especially 10⁸ TCID₅₀.

The pharmaceutical composition may generally include one or morepharmaceutically acceptable and/or approved carriers, additives,antibiotics, preservatives, adjuvants, diluents and/or stabilizers. Suchauxiliary substances can be water, saline, glycerol, ethanol, oil,wetting or emulsifying agents, pH buffering substances, or the like.Suitable carriers are typically large, slowly metabolized molecules suchas proteins, polysaccharides, polylactic acids, polyglycollic acids,polymeric amino acids, amino acid copolymers, lipid aggregates, or thelike.

For the preparation of vaccines, the recombinant poxvirus, preferablyMVA, according to the invention can be converted into a physiologicallyacceptable form. This can be done based on the experience in thepreparation of poxvirus vaccines used for vaccination against smallpox(as described by Stick) et al. 1974).

For example, the purified virus can be stored at −80° C. with a titre of5×10⁸ TCID₅₀/ml formulated in about 10 mM Tris, 140 mM NaCl pH 7.4. Forthe preparation of vaccine shots, e.g., 10²-10⁸ particles of the viruscan be lyophilized in 100 μl to 1 ml of phosphate-buffered saline (PBS)in the presence of 2% peptone and 1% human albumin in an ampoule,preferably a glass ampoule. Alternatively, the vaccine shots can beproduced by stepwise freeze-drying of the virus in a formulation. Thisformulation can contain additional additives such as mannitol, dextran,sugar, glycine, lactose or polyvinylpyrrolidone or other aids such asantioxidants or inert gas, stabilizers or recombinant proteins (e.g.human serum albumin) suitable for in vivo administration. The glassampoule is then sealed and can be stored between 4° C. and roomtemperature for several months. However, as long as no need exists theampoule is stored preferably at temperatures below −20° C.

For vaccination or therapy, the lyophilisate can be dissolved in anaqueous solution, preferably physiological saline or Tris buffer, andadministered either systemically or locally, i.e. parenteral,subcutaneous, intravenous, intramuscular, intranasal, or any other pathof administration know to the skilled practitioner. The mode ofadministration, the dose and the number of administrations can beoptimized by those skilled in the art in a known manner. However, mostcommonly a mammal, preferably a human, is vaccinated with a secondadministration about two weeks to six weeks after the first vaccinationadministration. Third, fourth, and subsequent administrations will mostcommonly be about two weeks to six weeks after the previousadministration.

The invention provides methods for immunizing mammals, including ahuman. In one embodiment a subject mammal, which includes rats, rabbits,mice, and humans are immunized comprising administering a dosage of arecombinant MVA to the mammal, preferably to a human. In one embodiment,the first dosage comprises 10⁸ TCID₅₀ of the recombinant MVA virus andthe second and additional dosages (i.e., third, fourth, fifth, etc.)comprise 10⁸ TCID₅₀ of the virus. The administrations can be in a first(priming) dose and a second, or further, (boosting) dose(s).

The immunization can be administered either systemically or locally,i.e. parenterally, subcutaneously, intravenously, intramuscularly,intranasally, or by any other path of administration known to theskilled practitioner.

CD8 T Cell and Antibody Responses

Immunizations with the recombinant MVA of the invention can induce arobust CD8 T cell response. In preferred embodiments, after the first,second, third, fourth, fifth, etc. immunization, the recombinant MVAinduces a robust CD8 T cell response in the mammal, preferably a human,against the encoded antigen that is greater than the CD8 T cell responseagainst the immunodominant viral CD8 T cell epitope, e.g. TSYKFESV (SEQID NO:5) encoded by the MVA vector. Preferably, after the second, third,fourth, fifth, etc. immunization, an immunodominant T cell response isinduced in the mammal, preferably a human, against the encoded antigen.Preferably, after the second, third, fourth, fifth, etc. immunization,the recombinant MVA induces a CD8 T cell response in the mammal,preferably a human, against the encoded antigen that is at least 10%,15%, 20%, 25%, 30%, or 35% of total CD8 T cells. Preferably, after thesecond, third, fourth, fifth, etc. immunization, the recombinant MVAincreases the CD8 T cell response in the mammal, preferably a human,against the encoded antigen at least 2-, 3-, 4-, 5-, or 10-fold (i.e.,from 1% to 2%, 3%, 4%, 5%, or 10% of total CD8 T cells) as compared tothe response with the encoded antigen after a single administration orincreases the CD8 T cell response in the mammal, preferably a human,against the encoded antigen at least 2-, 3-, 4-, 5-, or 10-fold ascompared to the T cell response of a viral antigen (e.g. B8R).Preferably, the recombinant MVA generates a CD8 T cell response in themammal, preferably a human, against the encoded antigen at least 2-, 3-,4-, 5-, or 10-fold as compared to the T cell response against a viralantigen (e.g. B8R) after a single administration. Most preferably, theCD8 T cell response in the mammal, preferably a human, against theencoded antigen increases with 2-, 3-, 4-, or 5-, etc. immunizations toa greater extent than the response against a viral late antigen (e.g.B8R).

The level of CD8 T cell response can be determined, for example, bycollecting approximately 100-120 μl of blood in FACS/heparin buffer.PBMCs can be prepared by lysing erythrocytes with RBC lysis buffer.PBMCs can then be co-stained in a single reaction for OVA- andB8R-specific CD8 T cells using an anti-CD8α-FITC, CD44-PerCPCy5.5 andMHC class I dextramers complexed with their respective H-2Kb bindingpeptides, SIINFEKL (SEQ ID NO:4) or TSYKFESV (SEQ ID NO:5). The MHCclass I SIINFEKL-dextramer (SEQ ID NO:4) can be labelled with PE and theTSYKFESV-dextramer (SEQ ID NO:5) with APC. Stained cells can be analyzedby flow cytometry on a BD Biosciences BD LSR II system. Ten thousandCD8+ T cells can be acquired per sample.

Alternatively, the level of CD8 T cell response can be determined bycollecting blood from an immunized mammal, preferably a human, andseparating peripheral blood mononuclear cells (PBMC). These can beresuspended in growth medium containing 5 μg/ml brefeldin A (BFA,“GolgiPlug”, BD Biosciences) with 1 μM of test peptides, includingpeptides against immunodominant MVA epitopes (i.e., TSYKFESV; SEQ IDNO:5) (“B8R”) and peptides derived from the expressed neoantigen. ThePBMC can then be incubated for 5 h at 37° C. in 5% CO2, harvested,resuspended in 3 ml cold PBS/10% FCS/2 mM EDTA and stored overnight at4° C. The following day, the PBMC can be stained with antibodiesanti-CD8a-Pac-Blue (clone 53-6.7), anti-CD62L-PE-Cy7,anti-CD44-APC-Alexa 750, and anti-CD4-PerCP-Cy5.5 (all antibodies fromBD Biosciences). The PBMC can be incubated with appropriate dilutions ofthe indicated antibodies for 30 min at 4° C. in the dark. After washing,cells can be fixed and permeabilized by using the Cytofix/Cytoperm™ Pluskit (BD Biosciences) according to the manufacturer's instructions. Afterwashing, PBMC can stained for intracellular interferon-γ (IFN-γ) using aFITC-conjugated anti-I FN-y antibody (BD biosciences) diluted inperm/wash buffer (BD Biosciences). Stained cells can be analysed by flowcytometry.

Immunizations with the recombinant MVA of the invention can induce arobust antibody response. Antibody responses can be measured by ELISA.

Within the context of this invention, a “robust CD8 T cell response”means a higher percentage of neoantigen-specific CD8 T cells than thepercentage generated with the same MVA construct containing the PrSpromoter (5′AAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAA 3′; SEQ ID NO:6) aftera single immunization. In some embodiments, the CD8 T cell responsedemonstrates at least 1.5-fold or 2-fold higher neoantigen-specific CD8T cells than that generated with the same MVA construct containing thePrS promoter (SEQ ID NO:6) after a single immunization.

Within the context of this invention, a “robust antibody response” meansan antibody titer that is greater than the antibody titer obtained withthe same MVA construct containing the PrS promoter (SEQ ID NO:6) after asingle immunization. In some embodiments, the antibody titer is at least1.5 fold or 2-fold greater than the antibody titer obtained with thesame MVA construct containing the PrS promoter (SEQ ID NO:6) after asingle immunization.

Whether a recombinant MVA induces a “robust CD8 T cell response” or a“robust antibody response” against a neoantigen can be determined asdescribed in the examples herein. For example, MVA13.5 short and MVA13.5long both induce a “robust CD8 T cell response” as herein defined.MVA13.5 long induces a “robust antibody response,” as herein defined.

Although the method preferably comprises a single administration of thevector, in some embodiments, two, three, four, five, six, seven, or moreimmunizations of a recombinant MVA can be administered to the mammal,preferably a human.

In preferred embodiments, the encoded antigen is a bacterial, viral, ortumor antigen. Preferably, the antigen is a foreign antigen to themammal, including a human.

Examples Example 1. Generation of MVA Recombinants

HeLa cells were infected with MVA-BN at an MOI of 10 (10 TCID₅₀ percell) and total RNA was prepared 2 and 8 hours post infection. Primersspecific for various MVA ORFs were generated and RACE-PCR (FirstChoice®RLM-RACE Kit, Life Technologies, Darmstadt, Germany) was used togenerate PCR products representative of the MVA RNAs encoding theseORFs. The PCR products were sequenced to identify the transcriptionstart sites. Based on this information, promoters were identified forthe RNAs encoding these ORFs. The MVA promoters for the following ORFswere inserted into MVA constructs (Baur et al., Journal of Virology,Vol. 84 (17): 8743-8752 (2010)) to drive expression of the ovalbumin(OVA) gene: MVA13.5 (CVA022; WR 018), MVA050L (E3L; WR 059), MVA022L(K1L; WR 032), and MVA170R (B3R; WR 185).

Example 2. Promoter-Dependent RNA Expression Levels In Vitro

Infection of Hela cells with MVA recombinant viruses at MOI of 10 wasdone using cold virus attachment on ice for 1 h. After attachment thecells were washed and the zero hour (0 h) time point was collected orcells were incubated at 37° C. for collection of other time points.Samples were collected at 0.5, 1, 2, 4, and 8 h p.i. Cells werehomogenized and total RNA was extracted. The RNA was DNAse digested andcDNA was synthesized using oligo(dT) priming. The resulting cDNApreparations were used as template in a Taqman based qPCR reaction forthe simultaneous amplification of OVA and actin cDNA. Samples were runin an AB7500 cycler from Applied Biosystem. The results are shown inFIG. 3.

Example 3. Promoter-Dependent Protein Expression Levels In Vitro

HeLa cells were cultured in DMEM with 10% FCS. Hela cells were infectedwith MOI of 10 (10 TCID₅₀ per cell) of the recombinant MVA virus.Infected cells were collected at 1, 2, 4, 6, 8, and 24 h p.i., fixed andpermeabilized. For each sample, half of the cells were stained for OVAprotein using a rabbit anti-chicken OVA antibody and the other half werestained for MVA antigens using a rabbit anti-VACV polyclonal antibody.Samples were analyzed using a FACSCalibur flow cytometry analyzer (BDBiosciences) and FlowJo software. The results are shown in FIG. 4.

Example 4. Mice Immunizations and Bleeds

Groups of mice (C57/B16) were used for the study. Each group received atotal of three immunizations. A PBS-injected group served as a controlfor immune responses. Blood was taken via the tail vein for analysis ofimmune responses throughout the study.

Mice were immunized i.p. with 10⁸ TCID₅₀ of the respective MVA virusesdiluted in PBS (300 μL, total volume) at weeks 0, 4 and 8. Bleeds for Tcell analysis were performed one week after each immunization and bleedsfor antibody analysis were performed three weeks after eachimmunization.

Example 5. T Cell Staining and Antibody Detection

Approximately 100-120 μl of blood per mouse was collected inFACS/heparin buffer. PBMCs were prepared by lysing erythrocytes with RBClysis buffer. PBMCs were then co-stained in a single reaction for OVA-and B8R-specific CD8 T cells using an anti-CD8α-FITC, CD44-PerCPCy5.5and MHC class I dextramers complexed with their respective H-2Kb bindingpeptides, SIINFEKL (SEQ ID NO:4) or TSYKFESV (SEQ ID NO:5). The MHCclass I SIINFEKL-dextramer (SEQ ID NO:4) was labelled with PE and theTSYKFESV-dextramer (SEQ ID NO:5) with APC. Stained cells were analyzedby flow cytometry on a BD Biosciences BD LSR II system. Ten thousandCD8+ T cells were acquired per sample. The results are shown in FIGS.5-6.

Serum from whole blood was prepared. Ovalbumin ELISA and MVA ELISA wereperformed to detect specific antibodies (Serazym kit of SeramunDiagnostika GmbH, Heidesee, Germany). The results are shown in FIG. 7.

We claim:
 1. A method of inducing a robust antibody response and/or arobust CD8 T-cell response against a neoantigen in a human comprisingadministering one or more administrations of a recombinant modifiedVaccinia Ankara (MVA) virus to the human; wherein the recombinant MVAcomprises a Pr13.5 promoter operably linked to a nucleotide sequenceencoding the neoantigen, wherein the Pr13.5 promoter comprises at least1 copy of a nucleic acid sequence of at least 40 bases having at least95% identity with SEQ ID NO:1.
 2. The method of claim 1, wherein thePr13.5 promoter comprises at least 1 copy of a nucleic acid sequencedescribed in FIGS. 8A-FIG. 8F.
 3. The method of claim 1, wherein thePr13.5 promoter comprises at least 1 copy of a nucleic acid sequencehaving at least 95% identity with SEQ ID NO:1.
 4. The method of claim 4,wherein the Pr13.5 promoter comprises at least 1 copy of a nucleic acidsequence having at least 98% identity with SEQ ID NO:1.
 5. The method ofclaim 4, wherein the Pr13.5 promoter comprises SEQ ID NO:1.
 6. Themethod of claim 1, wherein the Pr13.5 promoter comprises at least 1 copyof a second nucleotide sequence that has at least 95% identity with SEQID NO:1.
 7. The method of claim 1, wherein the Pr13.5 promoter comprisesat least 1 copy of a second nucleotide sequence described in FIGS.8A-8F.
 8. The method of claim 6, wherein the Pr13.5 promoter comprisesat least 1 copy of a second nucleotide sequence that comprises SEQ IDNO:
 1. 9. The method of claim 1, wherein the Pr13.5 promoter comprises anucleotide sequence that has at least 95% identity with SEQ ID NO:2. 10.The method of claim 9, wherein the Pr13.5 promoter comprises SEQ IDNO:2.
 11. A method of inducing an immunodominant T cell response againsta neoantigen in a human comprising administering one or moreadministrations of a recombinant modified Vaccinia Ankara (MVA) virus tothe human; wherein the recombinant MVA comprises a Pr13.5 promoteroperably linked to a nucleotide sequence encoding the neoantigen,wherein the Pr13.5 promoter comprises at least 1 copy of a nucleic acidsequence of at least 40 bases having at least 95% identity with SEQ IDNO:1; and wherein the immunodominant T cell response against theneoantigen is greater than a T cell response to a poxviral antigen. 12.The method of claim 11, wherein the immunodominant T cell response is atleast 2, 3, 4, or 5 fold greater than the T-cell response to thepoxviral antigen.
 13. The method of claim 11, wherein the poxviralantigen is B8R.
 14. The method of claim 11, wherein the Pr13.5 promotercomprises at least 1 copy of a nucleic acid sequence as described inFIGS. 8A-FIG. 8F.
 15. The method of claim 11, wherein the Pr13.5promoter comprises at least 1 copy of a nucleic acid sequence having atleast 95% identity with SEQ ID NO:1.
 16. The method of claim 11, whereinthe Pr13.5 promoter comprises at least 1 copy of a second nucleotidesequence that has at least 95% identity with SEQ ID NO:1.
 17. Arecombinant modified Vaccinia Ankara (MVA) virus comprising a Pr13.5promoter operably linked to a nucleotide sequence encoding a neoantigen,wherein the Pr13.5 promoter comprises at least 1 copy of a nucleic acidsequence of at least 40 bases having at least 95% identity with SEQ IDNO:1.
 18. The recombinant MVA of claim 17, wherein the Pr13.5 promotercomprises at least 1 copy of a nucleic acid sequence as described inFIGS. 8A-FIG. 8F.
 19. The recombinant MVA of claim 17, wherein thePr13.5 promoter comprises at least 1 copy of a nucleic acid sequencehaving at least 95% identity with SEQ ID NO:1.
 20. The recombinant MVAof claim 19, wherein the Pr13.5 promoter comprises at least 1 copy of anucleic acid sequence comprising SEQ ID NO:1.
 21. The recombinant MVA ofclaim 17, wherein the Pr13.5 promoter comprises at least 1 copy of asecond nucleotide sequence of at least 31 nucleotides that has at least95% identity with SEQ ID NO:1.
 22. The recombinant MVA of claim 17,wherein the Pr13.5 promoter comprises at least 1 copy of a secondnucleotide sequence comprising SEQ ID NO:1.
 23. The recombinant MVA ofclaim 17, wherein the Pr13.5 promoter comprises SEQ ID NO:2.